Hiv-1 vpr interactions with mitochondrial apoptosis inducing factor and methods of using the same

ABSTRACT

Assays to identify Vpr/AIF interaction and translocation inhibitors are disclosed.

FIELD OF THE INVENTION

The invention relates to the discovery that of HIV Vpr binds to AIF frommitochondria and to assays to identify modulators of the interaction.

BACKGROUND OF THE INVENTION

The HIV accessory protein Vpr has been identified as being capable ofcell cycle arrest and the induction of apoptosis. This observation isdescribed in PCT application PCT/US01/10028, which is incorporatedherein by reference.

The interaction of HIV Vpr with the human cellular protein hVIP has beendisclosed in PCT application PCT/US98/21432, which is incorporatedherein by reference.

Mitochondrial apoptosis inducing factor has been identified.

There is a need for drug discovery screens and novel drugs to prevent orinduce apoptosis and that are useful in the treatment of inflammatorydisease and cancer.

SUMMARY OF THE INVENTION

The present invention relates to methods of identifying compounds thatinhibit Vpr binding to AIF.

The present invention relates to methods of identifying compounds thatinhibit the nuclear translocation of Vpr in the presence of AIF.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It has been discovered that Vpr binds to AIF. It has been discoveredthat the complex formed by Vpr bound to AIF, which is normally found inthe mitochondria, translocates to the nucleus of cells.

AIF is known to induces apoptosis by a non-caspase 9 pathway. Vpr isknown to induce apoptosis by a caspase-9 pathway.

The discovery that Vpr binds to AIF provides the means to design anddiscover specific inhibitors. According to the present invention, Vprand AIF are used to used to screen compounds for specific inhibitors.Inhibitors are useful as anti-HIV agents as well as agents for thetreatment of inflammatory diseases and cancer. Purified Vpr and AIF maybe used in drug screens to identify compounds which dissociate thecomplexes and inhibit the formation of complexes. Compounds may also bescreened to identify those which inhibit the complex from translocatingto the nucleus.

One having ordinary skill in the art can isolate the nucleic acidmolecules that encode Vpr and AIF and insert them into expressionvectors using standard techniques and readily available startingmaterials. The coding sequence is operably linked to the necessaryregulatory sequences. Expression vectors are well known and readilyavailable. Examples of expression vectors include plasmids, phages,viral vectors and other nucleic acid molecules or nucleic acid moleculecontaining vehicles useful to transform host cells and facilitateexpression of coding sequences. The recombinant expression vectors ofthe invention are useful for transforming hosts which express theproteins

Host cells for use in well known recombinant expression systems forproduction of proteins are well known and readily available. Examples ofhost cells include bacteria cells such as E. coli, yeast cells such asS. cerevisiae, insect cells such as S. frugiperda, non-human mammaliantissue culture cells chinese hamster ovary (CHO) cells and human tissueculture cells such as HeLa cells.

In some embodiments, for example, one having ordinary skill in the artcan, using well known techniques, insert DNA molecules into acommercially available expression vector for use in well knownexpression systems. For example, the commercially available plasmidpSE420 (Invitrogen, San Diego, Calif.) may be used for production ofPAPA1 in E. coli. The commercially available plasmid pYES2 (Invitrogen,San Diego, Calif.) may, for example, be used for production in S.cerevisiae strains of yeast. The commercially available MAXBAC™ completebaculovirus expression system (Invitrogen, San Diego, Calif.) may, forexample, be used for production in insect cells. The commerciallyavailable plasmid pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) may,for example, be used for production in mammalian cells such as ChineseHamster Ovary cells. One having ordinary skill in the art can use thesecommercial expression vectors and systems or others to produce proteinsby routine techniques and readily available starting materials. (Seee.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed.Cold Spring Harbor Press (1989) which is incorporated herein byreference.) Thus, the desired proteins can be prepared in bothprokaryotic and eukaryotic systems, resulting in a spectrum of processedforms of the protein.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers, are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989).

The expression vector including the DNA that encodes either Vpr or AIFis used to transform the compatible host which is then cultured andmaintained under conditions wherein expression of the foreign DNA takesplace. The protein of the present invention thus produced is recoveredfrom the culture, either by lysing the cells or from the culture mediumas appropriate and known to those in the art. One having ordinary skillin the art can, using well known techniques, isolate the Vpr or AIF thatis produced using such expression systems. The methods of purifyingprotein from natural sources using antibodies which specifically bind tothe proteins may be equally applied to purifying proteins produced byrecombinant DNA methodology.

In addition to producing proteins by recombinant techniques, automatedpeptide synthesizers may also be employed to produce Vpr and/or AIF.Such techniques are well known to those having ordinary skill in the artand are useful if derivatives which have substitutions not provided forin DNA-encoded protein production.

Hybridomas which produce antibodies that bind to AIF or Vpr, and theantibodies themselves, are useful in the isolation and purification ofAIF or Vpr and protein complexes that include AIF and Vpr. In addition,antibodies are specific inhibitors of AIF or Vpr. Antibodies whichspecifically bind to the respective protein may be used to purify thatprotein from natural sources using well known techniques and readilyavailable starting materials. Such antibodies may also be used to purifythe protein from material present when producing the protein byrecombinant DNA methodology.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab fragments and F(ab)₂ fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies and humanized antibodies. Insome embodiments, the antibodies specifically bind to an epitope of AIFor Vpr. Antibodies that bind to an epitope is useful to isolate andpurify that protein from both natural sources or recombinant expressionsystems using well known techniques such as affinity chromatography.Such antibodies are useful to detect the presence of such protein in asample and to determine if cells are expressing the protein.

The production of antibodies and the protein structures of complete,intact antibodies, Fab fragments and F(ab)₂ fragments and theorganization of the genetic sequences that encode such molecules arewell known and are described, for example, in Harlow, E. and D. Lane(1988) ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. which is incorporated herein by reference.Briefly, for example, AIF, or an immunogenic fragment thereof, isinjected into mice. The spleen of the mouse is removed, the spleen cellsare isolated and fused with immortalized mouse cells. The hybrid cells,or hybridomas, are cultured and those cells which secrete antibodies areselected. The antibodies are analyzed and, if found to specifically bindto the protein, the hybridoma which produces them is cultured to producea continuous supply of antibodies. Antibodies against AIF arecommercially available from Chemicon International (Temecula, Calif.)catalog #AB16501.

The present invention relates to methods of identifying anti-HIVcompounds and compounds useful to treat cancer and autoimmune diseases.According to this aspect, the Vpr or a fragment of Vpr known to interactwith AIF is contacted with AIF or a fragment of AIF which interacts withVpr in the presence of a test compound. The affininty of the Vpr or afragment thereof to the AIF or fragment thereof is measured and comparedto the affinity of the Vpr or a fragment thereof to the AIF or fragmentthereof in the absence of a test compound. Compounds which can disruptthe binding of AIF to Vpr may be useful as anti-HIV compounds orcompounds useful to treat cancer and autoimmune diseases. An example ofa positive control in this drug screen assay would be anti-Vprantibodies which competitively bind to Vpr with respect to AIF. Anotherexample of a positive control in this drug screen assay would beanti-AIF antibodies which competitively bind to AIF with respect to Vpr.Such antibodies are useful as known compounds that disrupt the Vpr/AIFinteraction. Known quantities of Vpr and AIF may be combined underconditions suitable for binding. In some embodiments of the invention,the preferred concentration of test compound is between 1 μM and 500 μM.A preferred concentration is 10 μM to 100 μM. In some preferredembodiments, it is desirable to use a series of dilutions of testcompounds. In some aspects, a kit is provided for performing thismethod. The kit comprises a first container comprising HIV Vpr or afragment of Vpr known to interact with AIF; and a second containercomprising AIF or a fragment of AIF which interacts with Vpr.Optionally, the kit may further comprise instructions for performing thetest assay and/or a fourth container with a positive control such asanti-Vpr antibodies which competitively bind to Vpr with respect to AIFand/or anti-AIF antibodies which competitively bind to AIF with respectto Vpr.

Another aspect of the present invention relates to methods ofidentifying compounds that inhibit Vpr/AIF complexes from translocatingto the nucleus of a cell. Cells known to produce or exposed to Vpr arecontacted with test compounds. The level of Vpr and/or AIF in thenucleus and/or the level of Vpr and/or AIF in the cytoplasm is measuredand compared to the corresponding level from cells not contacted withthe test compound. In some embodiments of the invention, the preferredconcentration of test compound is between 1 μM and 500 μM. A preferredconcentration is 10 μM to 100 μM. In some preferred embodiments, it isdesirable to use a series of dilutions of test compounds.

EXAMPLE

The following sequences identified by accession number and referencesare incorporated herein by reference. VPR AJ404325 vpr, gag, pol, vif,vpu, env, and nef VPR AF316862 vif, vpr (Cameroon isolate) VPR AF325763vif, vpr (South African isolate) AIF XM 010246 also called “programmedcell death 8” or “PDCD8” AIF NM 004208

1. A method of identifying compounds that inhibit HIV Vpr binding to AIFcomprising a test assay that comprises the steps of: i) contacting a)HIV Vpr or a fragment of Vpr known to interact with AIF and b) AIF or afragment of AIF which interacts with Vpr in the presence of c) a testcompound and ii) comparing the level of HIV Vpr binding to AIF to thelevel of HIV Vpr binding to AIF in the absence of said test compound. 2.The method of claim 1 wherein a) HIV Vpr and b) AIF or a fragment of AIFwhich interacts with Vpr are contacted in the presence of c) a testcompound.
 3. The method of claim 1 wherein a) HIV Vpr or a fragment ofVpr known to interact with AIF and b) AIF are contacted in the presenceof c) a test compound.
 4. The method of claim 1 wherein a) HIV Vpr andb) AIF are contacted in the presence of c) a test compound.
 5. Themethod of claim 1 further comprising a positive control assay thatcomprises the steps of: i) contacting a) HIV Vpr or a fragment of Vprknown to interact with AIF and b) AIF or a fragment of AIF whichinteracts with Vpr in the presence of c) anti-Vpr antibodies whichcompetitively bind to Vpr with respect to AIF and/or anti-AIF antibodieswhich competitively bind to AIF with respect to Vpr.
 6. The method ofclaim 1 wherein the concentration of test compound is between 1 μM and500 μM.
 7. The method of claim 1 wherein the concentration of testcompound is between 10 μM to 100 μM.
 8. The method of claim 1 wherein aseries of test assays are performed using a series of dilutions of testcompounds.
 9. A kit for performing the method of claim 1 comprising: a)a first container comprising HIV Vpr or a fragment of Vpr known tointeract with AIF; and, b) a second container comprising AIF or afragment of AIF which interacts with Vpr in the presence of c) a testcompound; and optionally, c) instructions for performing the test assay.10. The kit of claim 9 further comprising a fourth container comprisinganti-Vpr antibodies which competitively bind to Vpr with respect to AIFand/or anti-AIF antibodies which competitively bind to AIF with respectto Vpr.
 11. A method of identifying compounds that inhibit HIV Vpr/AIFnuclear translocation comprising a test assay that comprises the stepsof: i) contacting, in the presence of a test compound, cells thatcomprise HIV Vpr or a fragment of Vpr known to interact with AIF and AIFor a fragment of AIF which interacts with Vpr in the presence and AIF;and ii) comparing the level of Vpr/AIF in the nucleus to the level ofVpr/AIF in the nucleus in the absence of said test compound and/orcomparing the level of Vpr/AIF in the cytoplasm to the level of Vpr/AIFin the cytoplasm in the absence of said test compound.
 12. The method ofclaim 10 wherein the cells comprise a) HIV Vpr and b) AIF or a fragmentof AIF which interacts with Vpr.
 13. The method of claim 10 wherein thecells comprise a) HIV Vpr or a fragment of Vpr known to interact withAIF and b) AIF.
 14. The method of claim 10 wherein the cells comprise a)HIV Vpr and b) AIF.
 15. The method of claim 1 wherein the concentrationof test compound is between 1 μM and 500 μM.
 16. The method of claim 1wherein the concentration of test compound is between 10 μM to 100 μM.17. The method of claim 1 wherein a series of test assays are performedusing a series of dilutions of test compounds.